Thursday, June 25, 2020

Media Components - Minerals, Chelators, Buffers, Antifoams, Oxygen.

Minerals

All microorganisms require certain mineral elements for growth and metabolism.  Magnesium, phosphorous, potassium, sulphur, calcium and chlorine are essential components to be added while media preparation.  The concentration of phosphate in a medium may be larger than the other media components, a part of this phosphate is used as a buffer to adjust pH variation during the fermentation process. Others such as cobalt, copper, iron, manganese, molybdenum and zinc are present in sufficient quantities in the water supplies and as impurities in other media ingredients. For example, corn steep liquor contains a wide range of minerals that will usually satisfy the minor and trace mineral needs.  Occasionally, levels of calcium, magnesium, phosphorous, potassium, sulphur and chloride ions are too low to fulfil requirements and these may be added as specific salts. Trace elements such as manganese, iron and zinc are reported to be important in secondary metabolite production.  

        Chelators

The insoluble metal phosphates form white precipitates while preparing or autoclaving many media. This can be avoided by the addition of chelating agents such as EDTA, citric acid, polyphosphates. They form complexes with metal ions such as iron, calcium, manganese and zinc. These can then be gradually utilised by microorganism. It is important to check the concentration of chelators otherwise it may inhibit the growth. In many media these are added separately after autoclaving. In large scale fermentations, complex ingredients such as crude industrial byproducts or yeast extract, peptone etc. complex with these metal ions ensuring their slow release.

        Buffers

The control of pH is extremely important if optimal productivity is to be achieved. pH variations occur during fermentation as metabolism proceeds and different acid/alkaline products are released in the media. This can further interfere with the growth and product formation. Buffers help to maintain the desired pH. A compound may be added to the medium to serve as a buffer and it may also be used as a nutrient source. Many media incorporate calcium carbonate to maintain the pH at 7.4. Carbonate will decompose if pH decreases. Phosphate added as a media component play an important role in buffering. However high phosphate concentrations can be critical in many secondary metabolite fermentations, so should be taken care of.

Buffering capacity can be provided by the balanced use of the carbon and nitrogen sources also such as corn steep liquor, peptone etc. which contains proteins, peptides and amino acids in addition to any contaminating carbon.  pH may also be controlled by the addition of ammonia or sodium hydroxide and sulphuric acid.

        Antifoams

Antifoams are necessary to reduce foam formation during fermentation.  Foaming is largely due to media proteins that become attached to the air-broth interface where they denature to form a stable foam “skin” that is not easily disrupted.  The foam may block air filters, resulting in the loss of aseptic conditions, if uncontrolled. The fermenter can become contaminated and microorganisms are released into the environment.  It is important to provide a headspace/ “freeboard” in fermenters to provide space for the foam generated. 

Foaming can be minimized by (i) the use of a defined medium and a modification of some of the physical parameters, e.g. pH, temperature, aeration and agitation (if the foam is due to media components), (ii) use of mechanical foam breakers (iii) addition of chemical antifoams. 

Antifoams are surface active agents that reduce the surface tension in the foams and destabilize the protein films or break the foam.

The ideal antifoam should have the following properties:

·         readily and rapidly dispersed with rapid action

·         high activity at low concentrations 

·         prolonged action

·         non-toxic to fermentation microorganisms, humans or animals

·         low cost

·         thermostability

·      compatibility with other media components and the process, i.e., having no effect on oxygen transfer rates or downstream processing operations

·         be heat sterilisable

            Natural antifoams include plant oils (e.g., from soya, sunflower and rapeseed), deodorized fish oil, mineral oils and *tallow. The synthetic antifoams are mostly silicon oils, poly alcohols and alkylated glycols.  Since antifoams are of low solubility, they need a carrier, e.g., *lard oil, liquid paraffin or castor oil, which may be metabolised.  The concentrations of many antifoams which are necessary to control foaming may reduce the oxygen transfer rate by as much as 50%.  Thus, antifoam addition should be kept to an absolute minimum.  Some antifoams may reduce the oxygen transfer rate as well as adversely affect downstream processing steps, especially membrane filtration.  If the oxygen transfer rate is too severely affected mechanical foam breakers may have to be considered.

*Tallow is fat  from beef

*lard fat from pork.

        Oxygen

Oxygen is very critical in fermentation as it can affect the growth rate and product formation. Depending on the amount of oxygen required by the organism, it may be supplied in the form of air containing about 21% (v/v) oxygen or occasionally as pure oxygen when requirements are particularly high.  The organism’s oxygen requirements may vary widely depending upon the carbon source.  The rate of metabolism of media components, the viscosity of the medium and the presence of antifoams (surface active agents and can interfere with oxygen transfer) all can influence the oxygen availability in a medium. For most fermentations the air or oxygen supply is filter sterilized prior to being injected into the fermenter. 

The specific oxygen uptake rate of a microorganism increases with increase in the dissolved oxygen concentration up to a certain point referred to as the critical level (Ccrit).  Maximum biomass production is achieved by maintaining the dissolved oxygen concentration greater than the critical level thus satisfying the organism’s maximum specific oxygen demand.  However, frequently the objective of a fermentation is to produce a product and not biomass. So, metabolic disturbance of the cell by oxygen starvation may enhance product formation.  

References

  1. Industrial Microbiology: An Introduction. Michael J. Waites, Neil L. Morgan, John S
  2. Principles of Fermentation Technology- Peter Stanbury, Allan Whitaker, Stephen Hall

 


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